DNA sequencing process using stable isotopes

• US 5,002,868 A
• Filed: 07/20/1988
• Issued: 03/26/1991
• Est. Priority Date: 07/20/1988
• Status: Expired due to Term

• Current Assignee: ATOM Sciences Corporation
• Original Assignee: ATOM Sciences Corporation
• Inventors: Jacobson, K. Bruce, Schmitt, Harold W.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/221,734
• Time in Patent Office: 979 Days
• Field of Search: 435/6, 435/91, 435/810, 436/501, 436/800, 436/803, 436/805, 536/27, 935/16, 935/17, 935/77, 935/78, 935/86, 935/88
• US Class Current: 436/94
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/803 Stabe free radicals, e.g. spin immunoassay
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A DNA sequencing process using specific stable isotopes associated with specific terminators for labels. The process includes a step of incorporating a stable isotope in at least one of the deoxynucleoside triphosphates and/or the dideoxynucleoside triphosphates such that a terminated strand has included within it or at the end a stable isotope such as an isotope of sulphur. Replicated strands are then separated by performing gel electrophoresis thereon. The location of the DNA strand with the stable isotope assigned to a terminator is analyzed preferably by resonance ionization spectroscopy. The stable isotopes can be chosen such that specific labels are assigned to at least one, and preferably to each base, in the dideoxynucleoside triphosphates. In the preferred embodiment, each of the bases (A, T, G and C) are associated with a specific stable isotopic label, and can be analyzed in a single track which enhances the accuracy of the sequencing process.

Claims

1. A DNA sequencing process using individual stable isotopes that are associated with individual deoxynucleotides and/or terminators comprising the steps of:

introducing template DNA strands with primer hybridization into a solution containing deoxynucleoside triphosphates (dNTP) having thymine, adenine, cytosine, and guanine bases;

DNA polymerase;

buffer;

magnesium ion; and

dideoxynucleoside triphosphate (ddNTP) wherein the ratio of dNTP to ddNTP is selected, said ddNTPs causing replication termination when incorporated into the replicated strand as dideoxynucleotide at a frequency dependent upon the ratio of dNTP to ddNTP;

incorporating a specific stable isotope into at least one of the dNTPs and or ddNTPs such that a terminated strand has a specific stable isotope associated with each terminator at its end;

separating the terminated strands by performing gel electrophoresis thereof; and

determining where each said specific stable isotope is located on the gel.

3. The DNA sequencing process of claim 1 wherein said specific stable isotope incorporated into at least one of the dNTPs and/or ddNTPs is a specific stable isotope of sulphur.

5. The DNA sequencing process of claim 1 wherein a separate specific stable sulphur isotope is incorporated into each type of dNTP and/or ddNTP such that each base type is labeled with a separate specific sulphur isotope.

8. The DNA sequencing process of claim 1 wherein said step of determining where each said specific stable isotope that was assigned to a given terminator has been localized in said gel comprises analyzing said synthesized and terminated strands with resonance ionization spectroscopy.

2. A DNA sequencing process using stable isotopes associated with deoxynucleotides and/or terminators comprising the steps of:

obtain DNA from a source of interest;

segregating a template DNA strand;

synthesizing a complementary strand of DNA by binding primer to the template strand;

introducing template DNA strands with primer hybridization into a solution containing deoxynucleoside triphosphates (dNTP) having thymine, adenine, cytosine, and guanine bases;

DNA polymerase;

buffer;

magnesium ion; and

dideoxynucleoside triphosphate (ddNTPs) wherein the ratio of dNTP to ddNTP is selected, said ddNTPs causing replication termination when incorporated into the replicated strand as dideoxynucleoside phosphate at a frequency dependent upon the ratio of dNTP to ddNTP;

incorporating a specific stable isotope of sulfur, wherein said specific isotope of sulphur is selected from a group consisting of ³² S, ³³ S, ³⁴ S and ³⁶ S, into at least one of the dNTPs and/or ddNTPs such that a terminated strand has a specific stable isotope of sulfur associated with the terminator at its end;

separating the terminated strands by performing gel electrophoresis thereof; and

determining where each said specific stable isotope of sulfur is located in the gel.

4. The DNA sequencing process of claim 2 wherein said specific stable isotope incorporated into at least one of the dNTPs and/or ddNTPs is a specific stable isotope of sulphur.

6. The DNA sequencing process of claim 2 wherein a separate specific stable sulphur isotope is incorporated into each type of dNTP and/or ddNTP such that each base type is labeled with a separate specific sulphur isotope.

7. The DNA sequencing process of claim 4 wherein said specific sulphur isotope is selected from a group consisting of ³² S, ³³ S, ³⁴ S and ³⁶ S.

9. The DNA sequencing process of claim 2 wherein said step of determining where each said specific stable isotope that was assigned to a given terminator has been localized in said gel comprises analyzing said synthesized and terminated strands with resonance ionization spectroscopy.